Red Blood Cell Deformability Research Articles

Pyruvate Kinase Thermostability Is Associated with Red Blood Cell Adhesion, Deformability and Oxygen Affinity in Patients with Sickle Cell Disease

Introduction: Sickle cell disease(SCD) is caused by a mutation in the beta globin gene that causes formation of abnormal hemoglobin S (HbS). Red blood cells (RBCs) from patients with SCD are less deformable and take on the shape of sickle upon deoxygenation. This results in multiple downstream effects including RBC adhesion, microvascular occlusion and chronic hemolysis. RBCs rely solely on anaerobic glycolysis to produce adenosine triphosphate (ATP), which is needed to maintain the cell's structure and function. Pyruvate kinase (PK) is a key enzyme involved in the last step of glycolysis. We have previously demonstrated a decrease in PK activity and stability as a novel pathophysiological feature of SCD. Currently there are several clinical trials ongoing investigating the efficacy of PK activation by small molecules such as mitapivat (NL8517, NCT04000165, NCT05031780), AG-946 (NCT04536792) and etavopivat (NCT04624659, NCT04987489). In this study we aim to investigate how PK properties correlate to sickle RBC properties in order to explore if impaired PK function is associated with specific features of the complex SCD pathophysiology. Methods: Homozygous HbS (HbSS) and HbS/ β0 thalassemia patients were eligible to participate. Patients who received blood transfusion (<3 months) were excluded. Dense RBCs (MCHC >41 g/dL, Advia 120/2120 Siemens) and HbF and HbS levels (Tosoh G8) were measured. PK and hexokinase (HK) activity were measured on purified RBCs. PK thermostability (reflected by % residual PK activity) was measured on purified RBCs lysates after one hour of incubation (53°C). RBC adhesion to laminin (Biolamina) was measured using a microfluidic device (IBIDI µ-Slide I 0.4). RBC hydration (Ohyper), RBC deformability (EImax) and point of sickling (PoS) were assessed by osmotic and oxygen gradient ektacytometry using the Laser-Optical Rotational Red Cell Analyzer (Lorrca, RR Mechatronics). Oxygen affinity (P50) was measured with the Hemox-Analyzer (TCS). Statistical analysis was performed in Graphpad Prism (Pearson's correlation). Results: Fifty-seven patients with SCD (53 HbSS, 4 HbS/β 0 thalassemia) were included of which 17 were children (median age 14 years [range 6-17]) and 40 were adults (median age 36 years [range 18-58]). Baseline characteristics are depicted in Table 1. Figure 1 shows a heatmap with correlation coefficients of all laboratory parameters from Table 1 as measured in this cohort. When focusing on PK properties we found that patients with SCD and lower PK thermostability had significantly higher absolute reticulocyte counts (ARC, p<0.0001), lower hemoglobin (p=0.012) and HbF (p=0.020) levels and increased RBC adhesion to laminin (p=0.007). Furthermore, PK thermostability was significantly correlated with EImax (p=0.017) and inversely correlated with PoS (p=0.011) and P50 (p=0.003), indicating that patients with less stable RBC PK had less deformable RBCs which sickled at a higher oxygen tension. RBC adhesion to laminin was significantly correlated with both ARC (p=0.0003) and dense RBCs (p=0.019). To explore whether our findings were a reflection of reticulocytosis we fractionated whole blood samples of HbSS patients according to cell density. We found that more RBCs from the most dense fraction adhered to laminin than from the less dense, reticulocyte enriched, fraction, even though RBCs from this less dense fraction had reduced PK-thermostability. Additionally, the most dense RBCs had the lowest PK activity, were less deformable and had a higher PoS (data not shown). Altogether these findings indicate that the reduced PK thermostability we found in our SCD cohort is associated with an overall functionally impaired RBC population with increased RBC adhesion and ARC. We found no correlation of markers of RBC dehydration (Ohyper and % dense RBCs) with PK thermostability which suggests that RBCs with reduced PK thermostability are not more dehydrated. Discussion/conclusion: This study shows for the first time a significant correlation between PK thermostability in sickle RBCs and RBC functions, such as adhesion, deformability and oxygen affinity. Our results suggest that enhancing the activity and stability of PK, with PK activators, is an attractive target in SCD that might improve other pathophysiological targets outside RBC metabolism. Future studies are needed to explore how activation of PK will affect these altered sickle RBC properties in vivo.

A Miniaturized, Rapid Technique for Measuring Red Blood Cell-Mediated Microcapillary Occlusion in HbAS, HbSC, and HbSS Sickle Cell Disease Genotypes

Introduction Sickle cell disease (SCD) is caused by a genetic mutation that produces sickle hemoglobin (HbS). HbS polymerizes upon deoxygenation, causing red blood cells (RBCs) to become sickle-shaped and poorly deformable. Stiff sickle RBCs contribute to microvascular occlusion and organ damage. Previous studies have developed microfluidic devices for assessing RBC-mediated occlusion under hypoxia, but the complexity of the setup and data acquisition limits their adoption. A rapid, easy-to-use, point-of-care assay for assessing RBC-mediated occlusion could increase throughput and aid in determining outcomes of novel therapies, including gene therapies, for SCD. Here, we induced RBC sickling using a chemical method in a simplified workflow to study hypoxic RBC-mediated microcapillary occlusion for healthy (HbAA), sickle cell trait (HbAS), hemoglobin SC disease (HbSC), and homozygous SCD (HbSS) RBCs. Additionally, a custom miniaturized impedance analyzer (MIA) was developed to provide a rapid and fully electronic measurement of occlusion within 10 minutes of sample perfusion. Methods Microfluidic devices were fabricated using standard photolithography and polydimethylsiloxane (PDMS) micro-molding protocols. Glass slides were coated with gold electrodes by sputter deposition and bound to the PDMS microfluidic devices using oxygen plasma. The microfluidic devices comprised six microcapillary arrays of widths 12, 10, 8, 6, 4, and 3 μm, with each array coupled with a pair of gold electrodes [3]. The MIA included an ADALM2000 software-defined instrument module, a custom-designed front-end interface board, and a Raspberry Pi single-board computing module. The ADALM2000 was used to generate and acquire a 10-kHz measurement signal, while the front-end interface board handled signal amplification and multiplexed signal routing through the microcapillary arrays. A custom graphical user interface on the Raspberry Pi allowed users to set experimental parameters and monitor data in real time. Venous blood samples were collected in EDTA tubes from participants with HbAS (n=9), HbSC (n=9), HbSS (n=13), and HbAA (n=6) under an IRB-approved protocol. To induce hypoxia, washed RBCs were suspended at 20% hematocrit in a 1.5% (w/v) sodium metabisulphite in 1× PBS buffer and incubated for 3 minutes at room temperature. For normoxia, RBCs from the same donors were suspended at 20% hematocrit in PBS and incubated with the same conditions. Samples were then perfused through the microfluidic device at a constant inlet pressure. After 10 minutes of perfusion, the change in electrical impedance across each array was used to calculate an occlusion index (OI), representing the percent occlusion of the capillary network. Results Normoxic OI for HbSS and HbSC was significantly higher than HbAA but there were no statistical differences in HbAA vs. HbAS and HbSC vs. HbSS (Figure 1A). HbAS, HbSC, and HbSS RBCs had significantly higher occlusion under hypoxia than in normoxia (Figure 1A, 1.46 ± 1.67% vs. 8.43 ± 2.78%, P = 0.004 for HbAS, 4.56 ± 2.77% vs. 27.1 ± 17.0%, P = 0.004 for HbSC, and 8.01 ± 6.26% vs. 58.3 ± 26.2%, P = 0.0002 for HbSS). Occlusion of HbAA RBCs remained low in both normoxia and hypoxia (2.70 ± 0.643% vs. 4.72 ± 0.953%, P = 0.0625). Hypoxic OI was significantly different between HbAA vs. HbAS (P = 0.017), HbAS vs. HbSC (P = 0.004), and HbSC vs. HbSS (P = 0.007). Hypoxic OI in HbSC and HbSS was associated with elevated markers of RBC hemolysis - absolute reticulocyte count (ARC, P = 0.0061) and lactate dehydrogenase (LDH, P < 0.0001, Figure 1B). Conclusion In normoxia, HbSC had comparable OI to HbSS likely due to HbC induced efflux of potassium ions and water resulting in cellular dehydration and poor RBC deformability. The hypoxic assay, however, unmasked the greater severity of HbSS. Further, elevated hypoxic OI in HbAS may be linked to higher risk of organ damage in this genotype. Chemically-induced hypoxia combined with electrical impedance-based measurement of RBC-mediated microcapillary occlusion offers a new technique for rapidly assessing RBC health and function in a low-oxygen environment. This approach eliminates the need for expensive, high-resolution microscopes and complex gas-exchange chambers. This may serve as a new standard for assessing the clinical efficacy of new treatments that improve RBC deformability and identifying at-risk patients in otherwise mild SCD genotypes.

Evaluation of Red Cell Deformability in Sickle Cell Disease Patients, Sickle Cell Carriers, and Healthy Controls Using Lorrca-Derived Parameters

BACKGROUND Red blood cell (RBC) deformability plays a critical role in RBC elongation and viscosity, and when reduced, perfusion of peripheral tissues and oxygen delivery are impaired. Intracellular ATP maintains water and ion homeostasis in RBCs; reduced ATP leads to water and ion loss, dehydration and loss of RBC elasticity. RBC dehydration increases mean corpuscular hemoglobin concentration (MCHC) and also affects distribution of RBC width (RDW). Patients with sickle cell disease (SCD) have reduced RBC ATP, and markedly reduced RBC deformability when compared to healthy individuals. Here, we employed Laser-Optical Rotational Red Cell Analyzer (LORRCA, RR Mechatronics) to evaluate deformability in RBCs from patients with SCD (HbSS) compared to RBCs from sickle cell carriers (HbAS) and ethnic-matched healthy controls (HbAA). We further analyzed how these LORRCA-derived parameters correlate with ATP, MCHC, and RDW. METHODS We enrolled adult subjects (age ≥ 18 years) of African-descent, and not recently transfused (within 8 weeks) under protocol NCT03685721 approved by the NHLBI Institutional Review Board. Fresh whole blood in EDTA was processed on the same day of collection. Cells were counted and diluted in polyvinylpyrrolidone (PVP) solution before undergoing LORRCA assays including deformability, osmoscan, and oxygenscan (HbSS subjects). The deformability assay uses the elongation Index (EI) to measure the cells' ability to undergo deformation with increasing shear stress at 0.95, 3, and 30 Pascals (Pa). The osmoscan assay measures the deformability under continuous osmotic changes (0-600mOsm/kg) and has several parameters: O min (RBC fragility); El max (maximum deformability and membrane flexibility); O max (osmolality where EI max is achieved) and O hyper (intracellular viscosity and maximum deformability in the hypertonic region). The oxygenscan assay measures the oxygen pressure at the time when RBCs start to become rigid (point of sickling). Whole blood levels of ATP were measured using LC-MS/MS with LLOQ at 50.0 μg/mL and converted to intracellular concentrations by dividing by the hematocrit (as a fraction). Descriptive statistics and correlation (Spearman) of ATP, RDW, and MCHC with each LORRCA parameter were performed using R (v4.2.3) and Prism(v9). RESULTS We studied 156 subjects which comprised of 63 HbSS (33 males), 61 HbAS (23 males) and 32 HbAA (8 males) genotypes. The average age in years (range) in HbSS was 32 (18-58); in HbAS, 43 (18-70), and in HbAA, 38 (19-72). As shear stress and osmotic pressure increased, the mean EI and osmolality were significantly (p<0.001, unpaired two-sample t-test) lower in the HbSS cohort compared to HbAS and HbAA (Figure 1). There was no significant difference in the mean EI and osmolality between HbAA and HbAS with increasing shear stresses and osmotic pressures. As shear stress increased, the correlation of deformability with reduced ATP got significantly stronger in the HbSS cohort (r= -0.351 at 0.95 Pa to r=-0.401 at 30 Pa) but the trend was reversed for the HbAS and HbAA cohort (Table 1). Under increasing osmotic pressure, a significant correlation was found between reduced ATP and maximal deformability (EI Max) in HbSS (p=0.008) compared to HbAS (p=0.896) and HbAA (p=0.114). MCHC was significantly correlated with RBC hydration state (O Hyper) in all 3 genotypic groups (HbSS, HbAS and HbAA). Similarly, RDW correlated with maximal deformability (EI Max) under increasing osmotic pressure in all 3 cohorts. In the HbSS subjects, there was a significant positive correlation of ATP and RDW with point of sickling (oxygenscan); MCHC was negatively correlated but was not significant. CONCLUSION RBC deformability was reduced in HbSS patients compared to HbAS and HbAA under increasing shear stress and osmotic pressure. ATP levels showed significant correlation with RBC deformability as measured by LORRCA, particularly in patients with SCD, suggesting increasing ATP levels as an important therapeutic strategy in patients with SCD. In keeping with published data, our analyses showed that RBC volume and size as well as intracellular hemoglobin concentration can affect deformability.

Functional and Morphological Characterization of RBCs Loaded with Factor IX Conjugated with an Anti-Glycophorin Ā Antibody

Red blood cells (RBCs) potentially can be used as drug carriers to improve pharmacokinetics (PK) for their unparalleled lifespan in circulation (120 days). Coupling factor IX (FIX) to the surface of RBCs is an innovative approach to boost FIX replacement therapy in hemophilia B. Here we studied effects of loading FIX to RBCs in vitro (morphology, rheological properties, procoagulant activity) and in vivo (PK of FIX-Ter119/RBC). To achieve RBC binding, purified human FIX was conjugated to the anti-mouse glycophorin A monoclonal antibody Ter119 and the FIX-Ter119 conjugates were separated and collected using SEC-HPLC. Then, mouse RBCs were incubated with either 10,000 or 50,000 molecules of FIX-Ter119 per one RBC, resulting in ~40% binding efficiency (~4,000 and ~20,000 molecules of FIX/RBC, respectively). Procoagulant activity of FIX/RBC was assessed in FIX-deficient human plasma using 3 assays: 1) activated partial thromboplastin time (aPTT), 2) rotational thromboelastometry (ROTEM), and 3) thrombin generation assay (TGA). FIX/RBC showed a dose-dependent activity in all the assays: i) aPTT was reduced (4,000 FIX/RBC: 57±1 s; 20,000 FIX/RBC: 48±1 s vs. control RBCs: 71±0.2 s, p<0.0001); ii) ROTEM: plasma clotting time was shortened and rate of clot strengthening was increased (45±6 and 69±3 vs 43±7 α, n.s. and p<0.001 vs. control, respectively) as was maximal clot firmness (maximum amplitude 19±3; 29±5 vs 22±3 mm, n.s. and p<0.05, respectively) between the RBCs bearing 4,000 and 20,000 FIX/RBC and control RBC, respectively (Figure 1A); iii) TGA: peak thrombin concentration was increased (134±18 and 193±17nM, for 4,000 and 20,000 FIX/RBC, respectively, p<0.05) vs. 74±27 nM for control RBCs (p<0.01). Altogether these results indicate that RBCs loaded with FIX-Ter119 possess specific FIXa activity, promote thrombin generation, and accelerate clotting in FIX-deficient plasma. Morphology and deformability of FIX/RBC were changed compared with control RBC. Scanning electron microscopy showed that the RBCs loaded with 4,000 and 20,000 FIX/RBC had increased echinocyte content (10±7% and 25±9%, respectively, p<0.01) while control RBCs contained only 3±1% echinocytes (p<0.001). After centrifugation at 1000g, the compressed RBCs had altered morphology, reflecting dissimilarity in cell deformability. The shape distribution for 4,000 and 20,000 FIX/RBC showed, respectively, 4±2% and 14±5% uncompressed biconcave RBCs, 8±3% and 12±8% intermediate mainly biconcave RBCs, 8±4% and 14±6% intermediate mainly polyhedral RBCs, and 63±20% and 70±24% fully compressed polyhedral RBCs (polyhedrocytes). In contrast, the unloaded centrifuged RBCs contained only 0.5±0.4% uncompressed biconcave RBCs, 1.5±0.6% intermediate mainly biconcave RBCs, 6±2% intermediate mainly polyhedral RBCs, and as much as 92±5% polyhedrocytes. Therefore, FIX/RBC demonstrated reduced deformability under mechanical compression caused by centrifugation. These results agree with ektacytometry data that showed a reduction in the deformability of RBC loaded with the FIX-Ter119 conjugates (maximum elongation index: 4,000 FIX/RBC: 0.40±0.09; 20,000 FIX/RBC: 0.34±0.04 vs 0.49±0.05 control RBCs, n.s. and p<0.01, respectively). Flow cytometry of FIX/RBC showed externalization of phosphatidylserine, but this increase was within normal levels (2.9±0.5% of phosphatidylserine-expressing RBCs). Agglutination assays showed that the FIX/RBC conjugates do not undergo agglutination at 4,000 FIX/RBC, but there was spontaneous agglutination of 20,000 FIX/RBCs, which limits the amount of FIX that can be loaded on the surface of RBC. Finally, we injected radiolabeled free FIX or FIX-Ter119 conjugates intravenously in mice to allow binding directly to circulating RBCs in vivo. The PK profile shows that more than 90% of the FIX-Ter119 conjugate was associated with RBCs and circulated stably bound to the RBCs for 24 hours, increasing the area under the PK curve 7.6 times vs. free FIX (Figure 1B). In conclusion: i) FIX can be loaded to the surface of RBCs, ii) FIX/RBC exert procoagulant activity, iii) FIX binding causes detectable, but apparently tolerable rheological changes in RBCs, and iv) FIX/RBC circulation time grossly exceeds that of free FIX. These preclinical studies support the potential utility of using FIX/RBC for treating bleeding conditions related to FIX deficiency.

RBC eNOS Transduces Regional O2 Gradients to Orchestrate RBC O2 Delivery Phenotype during Circulatory Transit

BACKGROUND: The role of endogenous endothelial NO synthase (eNOS) in mature RBCs is poorly understood. We have previously demonstrated functionally O2-dependent eNOS migration in RBCs, mediating (via s-nitrosylation) glycolytic metabolon assembly/disassembly during circulation in support of antioxidant systems during stress. We have further explored eNOS migration and now propose the following mechanism along with its functional significance. We (and others) suggest that hemoglobin (Hb) conformational change resulting from deoxygenation leads to its docking at the cytoplasmic domain of Band 3 (cdB3). We propose that (1) cdB3 consequently undergoes a conformational change that is transduced by Piezo1 (a mechanosensitive ion channel located in the RBC membrane); (2) Piezo1 activation results in calcium entry into the RBC, which (3) aids in the disassembly of the RBC cytoskeleton, (4) facilitates eNOS migration to the membrane (where calmodulin is mainly localized - thereby bringing these two important proteins together), and (5) binds to calmodulin, activating eNOS to generate nitric oxide (NO) - which then influences RBC phenotype that facilitates O 2 delivery, specifically related to RBC deformability and regulation of Hb~O 2 release, via enhanced Bohr effect. METHODS: Heparinized human, eNOS (-/-), or wt murine (C57BL/6J) red blood cells (RBCs) were imaged (Nikon) for the analysis of cytoskeletal protein arrangement and eNOS migration. RBCs were subjected to oxygenation/deoxygenation, with/without pre-treatment with the Piezo1 agonist Yoda1, or a membrane tension buffer grammostola mechanotoxin #4 (GSMTx4; decoupling Piezo1), or carbon monoxide (CO) to maintain R-state Hb conformation upon RBC deoxygenation. RBCs were fixed (paraformaldehyde 4%/glutaraldehyde 0.08%), permeabilized (Triton X100 amount 0.1%) and incubated with primary antibodies (spectrin, actin, protein 4.1, or eNOS), then with the secondary antibody prior to visualization. Interaction between eNOS and cytoskeleton proteins, as well as calmodulin, was determined using a proximity ligation assay (Duolink). Intracellular RBC calcium levels were assessed in oxygenated/deoxygenated RBCs utilizing an environmentally controlled fluorescent plate reader, and the calcium probe Fluo 3. NO production in RBCs was assessed using DAF-FM. To assess the functional significance of this mechanism, Hb-O 2 affinity was determined from murine blood (eNOS (-/-) or wt). Oxygen dissociation curves (ODCs), were constructed at 2 fixed CO 2 levels (thin film rotating tonometer; in conjunction with a gas blender; with samples measured by arterial blood gas machine).Bohr effect was assessed by determining the area between the ODCs (ABC) at the 2 fixed CO2 levels (~40mmHg and ~70mmHg). RESULTS: We observed several significant findings related to red RBC deoxygenation. Upon RBC deoxygenation, there is an increase in calcium levels indicated by the Fluo3 signal, which is Piezo1 dependent. Cytoskeleton proteins, such as Actin, Spectrin, and protein 4.1, undergo disassembly upon RBC deoxygenation. This disassembly process is contingent on both Piezo1-mediated calcium flux and the conformational changes in hemoglobin (Hb) (and not pO 2). We found that eNOS is associated with 4.1 throughout the oxy/deoxy cycle and that eNOS exhibits the same migratory pattern as cytoskeleton proteins to the RBC membrane, where calmodulin is located, irrespective of the RBC's oxygenation state. Additionally, when eNOS localizes in the membrane, it exhibits interaction with calmodulin. The generation of nitric oxide (NO) by eNOS, as indicated by the DAF-FM signal, increases in a manner dependent on both calcium, Piezo1 and Hb conformational transition. The functional consequence of this mechanism appears to be related to O 2 delivery phenotype during circulatory transit, with cell deformability being affected in addition to an inhibition of the Bohr effect in eNOS (-/-) compared to wt mice (2006 ± 61 vs 1892 ± 69 ABC in WT (n = 3) vs eNOS (-/-) (n = 4), respectively; p = 0.0747). SUMMARY: Upon circulatory transit, RBC deoxygenation triggers the activation of eNOS. This activated eNOS is associated with Piezo1-mediated calcium flux, which is facilitated by calmodulin. These events are coordinated with the migration of eNOS within the red blood cell between the cytosol and the cell membrane. The migration of eNOS is influenced by cytoskeleton proteins.

Evaluation of the Impact of a Naturally Occurring Beta Hemoglobin Variant, Hb G-Makassar, on Mature Red Blood Cell Function and Pathology in a Sickle Cell Disease Mouse Model

Background: Gene based therapy may provide a cure for individuals with sickle cell disease (SCD); base editing is a novel strategy with the ability to directly edit the mutation responsible for disease. The sickle hemoglobin (HbS) point mutation cannot be converted to wild-type HBB by an adenine base editor, however it can be converted to a naturally occurring hemoglobin variant, HbG-Makassar (HbG). HbG is a non-pathogenic, non-sickling hemoglobin variant. In vitro crystal structure analysis of HbG shows that it is highly similar to that of wildtype hemoglobin (HbA). However, few HbGG individuals (with no overt red cell disease) and no HbSG individuals have been identified; thus, the red blood cells (RBCs) of HbGG and HbSG individuals have not been evaluated functionally. The Makassar base editing strategy produces greater than 80% homozygous Hb-G product and has been evaluated in vitro, however erythroid culture of edited hematopoietic stem cells do not produce fully mature, enucleated red cells. Therefore, mouse models of HbGG and HbSG could be used to evaluate the function of mature, enucleated HbGG or HbSG RBCs. Methods: A humanized beta globin locus mouse (Townes) with the Makassar allele was created at by knocking-in the Makassar point mutation to generated WT/Makassar heterozygotes (HbAG) which were backcrossed to generated HbGG homozygotes. HbSG genotypes were subsequently generated by crossing to HbSS Townes mice. We analyzed the red cell function of HbAA, HbAS, HbSS, HbGG, and HbSG mice. Whole blood was collected through terminal enucleation. Complete blood counts were measured on a hematology analyzer (Heska and, ADVIA). Red cell deformability (elongation index, EI) was measured using oxygen gradient ektacytometry (LoRRCA) under normoxic (EImax) and hypoxic (EImin) conditions. Viscosity measurements were obtained on a Beckman cone and plate viscometer. Percent sickle cells were quantified through chemically induced sickling with sodium metabisulfite and counted by an individual blinded to genotype using light microscopy. Kidney, liver, and spleen were fixed, preserved in paraffin, stained using hematoxylin and eosin stains and imaged using a Keyence microscope. Flow cytometry was performed on bone marrow and whole blood to determine the erythroid maturation and mitochondrial retention of RBCs. Analysis was performed with Stata 18.0 (College Station) across genotypes using Dunn Pairwise test with statistically significant values being selected at adjusted p < 0.05. Results Homozygous HbG mice (HbGG) had comparable white blood cell count (WBC) and absolute reticulocyte count (ARC) to that of mice with homozygous HbA (HbAA). HbGG mice had lower hemoglobin and mean corpuscular volume (MCV) in comparison to mice with HbAA. HbGG mice had a higher percentage of dense RBCs than HbAA mice. HbSG mice had comparable values to HbAS mice for MCV but had significantly lower hemoglobin and ARC. HbSG mice had significantly higher values for WBC, and % dense RBCs against HbAS mice. HbSS and HbSG mice had comparable values for hemoglobin, and ARC. Hematocrit to viscosity ratio (HVR) shows comparable oxygen transport effectiveness of HbGG, HbSG, HbAS and HbSS RBC. Further red cell function testing is shown in Figure 1 and Figure 2. Mitochondrial retention was highest in HbSS mice, followed by HbSG, while HbAS, HbAA, and HbGG had similar values. Microscopy analysis of renal sections of HbGG and HbSG mice sacrificed at 24-28 weeks did not show the characteristic morphologic changes seen in HbSS mice at 24-28 weeks. Conclusions- HbGG appears functionally similar to HbAA, with minimal sickling under hypoxia, no anemia, normal spleen size, and no organ damage at 24 weeks of age. HbGG does exhibit signs of dehydration, as evidenced by elevated %DRBC and reduced RBC deformability. RBC function, sickling under hypoxia, Hb, WBC, ARC, and mitochondrial retention measures places HbSG as intermediate in severity between HbAS and HbSS. However, organ pathology, spleen and liver weights for HbSG were more comparable to HbAS than HbSS. Base editing strategies to install Makassar therefore can potentially lead to >90% of RBCs that have therapeutic benefit due to rheological properties comparable to HbAA and can be seen as a promising treatment strategy for patients with sickle cell disease.

Effect of vibration of the vortex mixer on the red blood cells

PurposesRed blood cells (RBCs) are often subject to vibration during processing, transfusion, and transport. Further research is necessary to understand the effects of vibration on human RBCs and to reduce experimental deviations caused by device vibration. MethodsFlow cytometry was used in this study to observe the cytokine expression of IgG and IgA and deformation of human red blood cells affected by the vibration of a vortex mixer with varying frequency (750 rpm and 1500 rpm), duration (5 min and 10 min), and container volume (96 well plate and 48 well plate). ResultsThe size of RBCs in duration of 10 min is obviously smaller than the duration of 5 min. The 10-minute duration led to visibly smaller RBC sizes compared to the 5-minute duration. There was little effect on the size of RBCs in the 10-minute groups from differences in frequency and container volume. However, decreased RBC size can be observed in the 5-minute groups, where frequency is increased or container volume is decreased. Echinocytes were present in photomicrographs of all 10-minute groups, but microstructure of the RBCs was not impacted by vortex mixer vibration. The elevated frequency or reduced container volume results in an increased cytokine expression of IgG within the 5-minute groupings. ConclusionIt can be inferred that vibration must not be overlooked due to its potential impact on the shape and cytokine expression of RBCs. Hence, the inclusion of vibration must be taken into consideration in experiments and devices pertaining to RBCs.

Quantifying the deformability of malaria-infected red blood cells using deep learning trained on synthetic cells

Quantifying the deformability of malaria-infected red blood cells using deep learning trained on synthetic cells

High-throughput quantification of red blood cell deformability and oxygen saturation to probe mechanisms of sickle cell disease.

The complex, systemic pathology of sickle cell disease is driven by multiple mechanisms including red blood cells (RBCs) stiffened by polymerized fibers of deoxygenated sickle hemoglobin. A critical step toward understanding the pathologic role of polymer-containing RBCs is quantifying the biophysical changes in these cells in physiologically relevant oxygen environments. We have developed a microfluidic platform capable of simultaneously measuring single RBC deformability and oxygen saturation under controlled oxygen and shear stress. We found that RBCs with detectable amounts of polymer have decreased oxygen affinity and decreased deformability. Surprisingly, the deformability of the polymer-containing cells is oxygen-independent, while the fraction of these cells increases as oxygen decreases. We also find that some fraction of these cells is present at most physiologic oxygen tensions, suggesting a role for these cells in the systemic pathologies. Additionally, the ability to measure these pathological cells should provide clearer targets for evaluating therapies.

Oxygen gradient ektacytometry–derived biomarkers are associated with acute complications in sickle cell disease

AbstractWe investigated the potential of the point of sickling (PoS; the pO2 tension at which red cells start to sickle), determined by oxygen gradient ektacytometry to serve as a biomarker associated with the incidence of acute sickle cell disease–related complications in 177 children and 50 adults. In the pediatric cohort, for every 10 mmHg increase in PoS reflecting a greater likelihood of sickling, the likelihood of an individual experiencing >1 type of acute complication increased; the adjusted odds ratio (aOR) was 1.65. For every 0.1 increase in minimum elongation index (EImin; reflecting improved red blood cell deformability at hypoxia), the aOR was 0.50. In the adult cohort, for every 10 mmHg increase in PoS, we found an aOR of 3.00, although this was not significant after correcting for multiple testing. There was a trend for an association between higher PoS and greater likelihood of vaso-occlusive episodes (VOEs; children aOR, 1.35; adults aOR, 2.22). In children, only EImin was associated with VOEs (aOR, 0.68). When data of both cohorts were pooled, significant associations with PoS and/or EImin were found for all acute complications, independently and when >1 type of acute complication was assessed. These findings indicate that oxygen gradient ektacytometry generates novel biomarkers and provides a rationale for further development of these biomarkers in the assessment of clinical severity, evaluation of novel therapies, and as surrogate clinical trial end points. These biomarkers may be useful in assessing efficacy of novel therapies like pyruvate kinase activators, voxelotor, and L-glutamine.

Effect of sodium nitroprusside on the microrheological properties of red blood cells in different media

Red blood cell (RBC) aggregation as well as their deformation significantly affects blood microrheology. These processes depend on various factors, one of which is concentration of the nitric oxide, one of the main signaling molecule in the bloodstream. The purpose of this study was to investigate the effect of nitric oxide on the microrheological properties of red blood cells (RBCs) in RBC samples of various media after the addition of nitric oxide donor sodium nitroprusside in vitro. Microrheological properties were measured using laser aggregometer and ektacytometer based on diffuse light scattering and diffraction of laser light on a suspension of RBCs, respectively. The study found that heparin-stabilized blood showed increased RBC aggregation and deformation with sodium nitroprusside concentrations of 100, and 200[Formula: see text][Formula: see text]M, while EDTA-stabilized blood showed slightly decreased aggregation and unchanged deformation. With washed RBCs in dextran solution, the addition of sodium nitroprusside (in the concentrations of 100, and 200[Formula: see text][Formula: see text]M) resulted in decreased aggregation and increased deformation. These findings aid in our understanding of nitric oxide’s effect on RBC microrheological properties.

Enhancing Adaptive Physics Refinement Simulations Through the Addition of Realistic Red Blood Cell Counts.

Simulations of cancer cell transport require accurately modeling mm-scale and longer trajectories through a circulatory system containing trillions of deformable red blood cells, whose intercellular interactions require submicron fidelity. Using a hybrid CPU-GPU approach, we extend the advanced physics refinement (APR) method to couple a finely-resolved region of explicitly-modeled red blood cells to a coarsely-resolved bulk fluid domain. We further develop algorithms that: capture the dynamics at the interface of differing viscosities, maintain hematocrit within the cell-filled volume, and move the finely-resolved region and encapsulated cells while tracking an individual cancer cell. Comparison to a fully-resolved fluid-structure interaction model is presented for verification. Finally, we use the advanced APR method to simulate cancer cell transport over a mm-scale distance while maintaining a local region of RBCs, using a fraction of the computational power required to run a fully-resolved model.

Novel RBC Adhesion and Deformability Assays Reveal Deleterious Effect of Diabetes on RBC Health

Diabetes is one of the most important health issues in the world. The number of people living with diabetes has quadrupled since 1980 1. Cardiovascular disease (CVD), mainly coronary and artery disease, remains the most common cause for morbidity and mortality in people living with diabetes 2,3. A better understanding of the effect of diabetes on the deformability and adhesiveness of red blood cells (RBCs) is essential in assessing disease status and evaluating the risk of CVD, as well as in investigating the effectiveness of potential therapies 4-6. In this work we investigate the deformability of RBCs and their adhesion to erythrocytes using two proprietary microfluidic assays, OcclusionChip 7 and Adhesion Biochip 8 respectively. We previously developed these assays for investigating RBC properties. Occlusion chip assay, consisting of micropillar arrays ranging from 20 µm spacing to 4 µm spacing along the flow direction, creating a gradient of microcapillaries, were fabricated using soft lithography and perfused with isolated RBCs at a constant inlet pressure. Venous samples from diabetic or non-diabetic individuals were collected in EDTA tubes and tested within 48 hours of collection. RBCs were isolated from whole blood and resuspended in PBS at 20% hematocrit, then flown through the chip using a pressure pump for 20 min. The microchannel was then washed and imaged using an inverted microscope. The cells trapped within the micropillar arrays were manually counted to calculate the occlusion index, which we previously established as a biomarker for cell deformability 9. To quantify RBC adhesion to endothelial cells, human umbilical vein endothelial cells (HUVECs) were cultured within a microfluidic channel under flow for 48 h. HUVECs were then activated by heme at a concentration of 40 µM for 1 h at 37 °C. Isolated RBCs resuspended in basal cell culture medium at 20% hematocrit supplemented by 10 mM of HEPES were injected in the microchannel for 15 min. Non-adherent RBCs were rinsed with a basal cell culture medium containing HEPES. The remaining adherent erythrocytes were imaged using an inverted microscope and manually quantified. Our results show that RBCs isolated from individuals with diabetes (A1c > 6.5%) tend to form more occlusion events (OI) than RBCs collected from healthy individuals (A1c < 5.7%), reflecting a loss of deformability (Fig. 1). For comparison we evaluated the elongation index (EI) using ektacytometry (data not shown), and no statistically significant difference was observed. Moreover, RBCs with diabetes tend to be more adhesive to the endothelium than those from healthy individuals, as evident from the count of adherent RBCs (Fig. 2). In conclusion, our assays revealed that diabetes presents a significant adverse effect on the deformability and adhesion of RBCs. These assays provide a much-needed tool for the quantification of these effects for the assessment of CVD risk and the evaluation of treatment efficacy. Future work will focus on conducting a larger-scale study to confirm our preliminary results. We will test samples from patients in all clinically relevant ranges of disease status, i.e. healthy, pre-diabetes, and diabetes.

Roxyscan: A Novel Method to Assess Red Blood Cell Resilience to Oxidative Stress in Sickle Cell Disease

Background: Red blood cells (RBCs) are biconcave-shaped cells with viscoelastic membranes that are optimally adapted for oxygen delivery and gas exchange during their normal lifespan of 120 days. Reactive oxygen species (ROS) continuously challenge the viability of RBCs. Despite an arsenal of compounds to protect the RBC, including Glutathione (GSH), Vitamin E, superoxide dismutase (SOD) and Catalase, damage can easily be inflicted that will disrupt cellular deformability, structure, and membrane properties. The susceptibility to oxidative stress in individuals with Sickle Cell Disease (SCD) is believed to be linked to multiple factors. These include the release of heme and iron during hemolysis, inflammation processes, and a reduction in antioxidant defenses. Methods:Susceptibility of RBCs to oxidative stress was assessed with the RoxyScan. This novel application of the Lorrca MaxSis (RR Mechatronics) measures rheological behavior of RBCs in response to cumene hydroperoxide (CuHP, 100 µM) exposure during continuous shear (30 Pa). For this we used the Lorrca's peristaltic pump and suction needle, directing the blood and CuHP to the instrument's cup and mixing them for 30 seconds (s) before starting the run. Deformability of RBCs (EI, elongation index) was measured over time. PoX is defined as the time where a 10% decrease in EI is reached. EI start: EI t=0. EI final: EI t=1600s, ΔEI (EIstart-EIfinal). RoxyScan measurements were performed on blood samples from 16 adult SCD patients (HbSS, HbS/β0 or HbS/β+) and 19 healthy controls (HC). Outcome measurement PoX (calculated from curve fitted data) was correlated to laboratory parameters from complete blood count (Cell Dyn Sapphire, Abbott), RBC sickling tendency (Point of Sickling, PoS, as assessed by oxygen gradient ektacytometry on the Lorrca MaxSis), and levels of HbF/HbS (Tosoh G8). Results:RBCs from untreated HC show a stable EI over time (Fig 1A). Upon exposure to CuHP a similar decrease in EI is seen in 19 HC (Fig 1A), mean decrease 0.055, range (0.017 to 0.10). The mean and median calculated PoX in HC is 1723s and 1702s respectively. In contrast, SCD RBC showed an earlier and more pronounced decrease in EI in response to CuHP exposure (representative example, Fig 1A). Comparing the PoX between HC and SCD revealed significant differences: mean PoX HC: 1723s (SD 329s), SCD: 725s (SD 472s), ***p<0.0001, indicating that SCD RBCs are more susceptible to oxidative stress. Correlating PoX to routine laboratory parameters identified a correlation with Hb levels (***p=0.002), inverse correlation with reticulocyte count (***p=0.0014) and hemoglobin distribution width (HDW, ***p<0.001) (Fig 1B). Similarly strong inverse correlations were found for EI start and EI end (***p<0.001) (Fig 1B). PoX also showed an inverse correlation with red cell distribution width (RDW, ns p=0.07) and mean corpuscular volume (MCV, ns p=0.77). Interestingly, PoX showed a significant inverse correlation with PoS (***p=0.004), indicating that SCD RBCs which are more susceptible to oxidative stress tend to sickle at a higher oxygen tension. PoX showed no significant correlations with HbS/HbF levels (Fig 1B). Conclusion: In this study we demonstrate the applicability of the RoxyScan as a novel method to assess RBC resilience to oxidative stress. When applied to SCD, RBCs show an earlier and more pronounced response to oxidative stress than RBCs from HC. The inverse correlation of PoX, the major outcome parameter of this technique, with reticulocyte count could indicate a generally increased susceptibility to oxidative stress due to a higher level of hemolysis. The correlation with Hb levels affirms this. PoX showed other strong and significant correlations with routine laboratory parameters, such as HDW, implying that the greater variability in hemoglobin content is associated with higher susceptibility to oxidative stress. On a functional level, it is interesting to find strong correlations of PoX with PoS, showing a direct link between RBC sickling tendency and ability to withstand oxidative stress. On the other hand, HbS and HbF levels did not correlate significantly to PoX. Overall, our study highlights the potential use of the RoxyScan and its associated biomarkers in the analysis of the RBC's response to oxidative stress. Further research is in progress to investigate the clinical applicability of this new technique.

Biologic Assessment of RBC Biology and Neutrophil Activation: Correlation with Sickle Cell Disease Activity

Sickle cell disease (SCD) has pleiomorphic effects on affected individuals, most typically including both hemolysis and vaso-occlusive events (VOE). However, previous reports have suggested that these two paradigmatic presentations of SCD represent two poles of a spectrum of disease activity, so that patients with the highest frequency of VOEs are at one end, and patients with high hemolytic rates and organ damage, such as nephropathy and pulmonary hypertension, are at the other end. We performed an observational study of erythroid (RBC) factors, neutrophil activation, and clinical parameters, including vaso-occlusion event (VOE) frequency, in order to determine which factors were related to each other and to either hemolytic rate or VOE frequency. 115 patients in steady state, at least 3 weeks after last VOE, with either HbSS, HbSß +/0 thalassemia, or HbS/HPFH, were enrolled from 10/20/2020 - 10/25/2022. Erythrocytes and plasma were isolated from whole blood and analyzed for RBC deformability, intracellular RBC [ATP], RBC phosphatidylserine exposure, and plasma matrix metallopeptidase 9 (MMP9), a product of neutrophil activation and degranulation. A subset of samples were examined for RBC reactive oxygen species (ROS) content and adhesivity to laminin with and without stimulation by epinephrine. Clinical data collected included complete blood count parameters, LDH, total bilirubin, reticulocyte (retic) counts, and health care utilization for VOE in the preceding year. Deformability was measured as maximal elongation index (EImax) at Pa=30 by laser optical rotational red cell analyzer (Lorrca). RBC ROS was quantitated using 2′,7′-dichlorofluorescein and expressed as nM/mg Hb. MMP9 was measured by commercial ELISA assay. We found (N=102 with complete data) that EImax was directly proportional to total [Hb] (P<0.0001), irrespective of gender. Higher EImax was also associated with lower parameters of hemolysis (P = <0.0001, 0.0002, 0.0004, and <0.0001 for total LDH, total bilirubin, absolute retic count, and % retics, respectively). Higher HbF levels were associated with greater deformability (P<0.0001), and RBC deformability was inversely associated with PS exposure (P<0.0001) and RBC ROS content (P=0.0484, N=23). Surprisingly, however, greater deformability was not associated with higher RBC ATP content or lower health care utilization for VOE, while there was only a trend toward an inverse association with plasma MMP9 (P=0.0771). Interestingly, we also found that RBC [ATP] was lower in the RBCs of patients with higher total Hb, while it was higher in individuals with higher retic counts, suggesting that more long-lived RBCs have diminished [ATP], despite the fact that both [ATP] and total Hb have been reported to rise substantially after treatment with pyruvate kinase activators etavopivat and mitapivat. Higher ATP was also not significantly associated with a decrease in RBC adhesivity. Of note, higher HbF levels were significantly associated with lower hospital admissions for VOE (P=0.0053), although not with frequency of out-patient health care utilization for VOE (ED and day hospital visits). HbF levels also showed a trend toward inverse association with plasma MMP9 (P=0.0613). These results may help explain why therapeutic agents that increase total Hb by shifting the oxygen dissociation curve, thereby reducing sickling and hemolysis, appear to have little or no effect on VOE rate. Similarly, senicapoc, which also reduces hemolysis, although by a different mechanism, failed to lessen VOE frequency. We conclude that RBC deformability, PS exposure, and RBC ROS are markers associated with the hemolytic subphenotype but not with the somewhat overlapping but divergent adhesiveness and frequent VOE subphenotype. Anti-adhesive agents (crizanlizumab, rivipansel, sevuparin) have thus far failed to yield marked improvements in VOE frequency, but our results suggest that, in order to decrease the frequency of VOEs, it is not sufficient to reduce sickling and hemolysis. On the other hand, higher HbF levels were associated with lower admission rates for VOE and a trend toward lower levels of neutrophil activation. This suggests that higher HbF, beyond its ability to reduce sickling, also alters other biological characteristics of SS RBCs and is accompanied by reduction in the inflammatory nature of SCD, thereby contributing to a decrease in VOE frequency.

Voxelotor and Red Blood Cell Pyruvate Kinase Activator Affect Clot Strength in Sickle Cell Disease

Introduction: Sickle cell disease (SCD) is a common genetic red blood cell (RBC) disorder that results from a single substitution of valine for glutamic acid in the β-globin gene, resulting in sickle hemoglobin (HbSS). Under hypoxia, HbS polymerizes and forms fibers within the RBCs, thus making the cells rigid, deformed, and sickle-shaped [1]. Deformed RBCs influence the compactness and dynamics of blood clots [2]. In addition, people with SCD are susceptible to thrombophilia [2,3,4]. Recently, there has been a heightened interest in ameliorating SCD using RBCs pyruvate kinase (PKR) activators, which increase ATP and decrease 2,3 DPG, resulting in increased oxygen affinity and improved RBCs deformability [5]. Voxelotor is also an oxygen affinity-modifying drug, recently FDA-approved. Voxelotor delays deoxy-HbSS formation, preventing in vitro HbSS polymerization and RBC sickling, thus improving RBC deformability [6]. The in vitro impact of PKR activators and Voxelotor on blood clotting in clinical samples from people with SCD has not been examined. Here we use a microfluidic dielectric sensor, termed ClotChip ® [7,8], to investigate the effects of Voxelotor and a PKR activator on the clotting time and clot strength of samples from people with SCD. Methods: Venous blood samples were collected in sodium-citrate from subjects with homozygous HbSS and healthy volunteers (HbAA) under an IRB-approved protocol. ClotChip ® microsensors were fabricated as previously described [7]. Blood samples were centrifuged at 200 g for 10 min. Plasma and buffy coats were removed and stored at 4 °Cfor 6 hours. Isolated, washed RBCs were then re-suspended in PBS at 20% hematocrit and then mixed with 67 mg/mL of Voxelotor (GBT440; Selleckchem) to a final concentration of 600 µM (in DMSO) or a PKR activator (PKR activator 3; MedChem Express) at 10 mM (in DMSO) and incubated at 37 °Cfor 6 hours. For controls, HbSS- or HbAA-containing RBCs at 20% hematocrit were mixed with PBS containing 0.5% v/v DMSO and incubated at 37 °C for 6 hours. After incubation, HbSS+Vox, HbSS+PKR, and control samples were centrifuged at 500 g for 10 min to remove PBS. Each sample's plasma was used to reconstitute RBCs at 20% hematocrit. CaCl 2 was added to the samples to induce coagulation, and the samples were injected into the ClotChip ® microfluidic sensors. An Agilent 4294A impedance analyzer was used to obtain the ClotChip ® readout curve defined as the temporal variation of the normalized real part of blood dielectric permittivity at 1 MHz (see Figure 1A). Based on our previous studies [7,8], the time to reach a permittivity peak (T peak) parameter was taken to indicate the clotting time, whereas the maximum change in permittivity after the peak (ΔƐ r,max) parameter was taken to indicate the clot strength. Data are reported as mean ± standard deviation (SD). Results: We analyzed the ClotChip ® T peak and ΔƐ r,max readout parameters for HbSS (n=11), HbSS+Vox (n=11), HbSS+PKR (n=11), and HbAA control (n=8) samples ( Figure 1B). The clot strength was ~50% higher in HbAA than HbSS (HbAA vs. HbSS: p= 0.001, Mann-Whitney test). However, when the SCD samples were treated with Voxelotor, the clot strength was observed to increase by ~10% (HbSS vs. HbSS+Vox, p=0.01, Paired t-test). Treatment with a PKR activator increased the clot strength by ~30% (HbSS vs. HbSS+PKR, p=0.003, Figure 1B). A significant difference was observed in the clotting time between HbAA vs. HbSS (p=0.001) but treatment with Voxelotor or a PKR activator did not change the clotting time for HbSS (HbSS vs. HbSS+Vox, p=0.43; HbSS vs. HbSS+ PKR, p=0.29) ( Figure 1B). (p values were determined using a non-parametric Mann-Whitney test and paired t-test). Discussion : We found that treatment of HbS-containing RBCs with a PKR activator or voxelotor improves clot strength but not clotting time. PKR activators improved the strength of the clot significantly more than Voxelotor. Patients with SCD had a faster clotting time than healthy participants, which agrees with earlier researchers on hypercoagulability in SCD [4]. These data suggest that HbS polymerization and RBC health affect clot characteristics but not clot kinetics in SCD. Further investigations may be required to relate RBC deformability, which is well-known to impact SCD pathophysiology, to blood clotting and venous thromboembolism.

Effects of L-Glutamine on Biomarkers of Response in Sickle Cell Disease: A Pharmacokinetics-Pharmacodynamics Analysis

OralL-glutamine (Endari®) has been approved by the US-FDA to reduce the acute complications of sickle cell disease (SCD) in patients at least 5 years of age. L-glutamine has several roles in the body including de novo synthesis of intracellular glutathione which acts as an antioxidant. However, the mechanisms of action by which L-glutamine could reduce complications in SCD require further investigation. Additionally, there are no known biomarkers to assess response to L-glutamine therapy. We conducted an open-label, dose-ascending trial (NCT04684381) of L-glutamine in pediatric (n=4) and adult (n=4) participants with SCD as well as in adult healthy volunteers (n=4) (Table 1). Over a three-week trial period with 4 study visits, serial blood samples were collected to define the pharmacokinetics (PK), pharmacodynamics (PD), and PK-PD interactions of L-glutamine using a broad panel of laboratory investigations including amino acid concentrations, blood counts, percentage of dense red blood cells (%DRBC), whole blood viscosity, osmotic and oxygen gradient ektacytometry, and reactive oxygen species (ROS) of reticulocytes (ROS retic) and mature red blood cells (ROS RBC). In PK-PD analysis of amino acids, we focused analysis on arginine (Arg), citrulline (Cit), and ornithine (Orn) based on the hypothesis that L-glutamine improves endothelial function by increasing arginine bioavailability and augmenting nitric oxide (NO) production. Peak arginine concentration was directly correlated with both the maximum L-glutamine concentration (C max p=0.001) and area-under-the curve (AUC) (p=0.047) indicating improvement in arginine bioavailability, but there was no significant linear correlation with either the Arg:Orn or Arg:(Orn+Cit) ratios. In PK-PD analysis of osmotic gradient ektacytometry, L-glutamine C max was directly correlated with the elongation maximum (EI max), noted in all participants at visit 4 (p=0.033). In SCD participants, L-glutamine C max was inversely correlated with O hyper (osmolality at EI max/2) at visits 3 (p=0.044) and 4 (p=0.004). In PK-PD analysis of oxygen gradient ektacytometry in SCD participants, L-glutamine C max was inversely correlated with the point of sickling (POS) at visit 3 (p=0.005) but not at visit 4 (p=0.054). Together, these findings suggest that although L-glutamine may decrease cellular hydration (decreased O hyper) it may increase RBC deformability (increased EI max) and delay the onset of sickling after hypoxia (decreased POS). In PK-PD analysis of viscometry, higher L-glutamine C max was associated with higher whole blood viscosity measurements at all shear rates (p<0.05). However, L-glutamine C max was also directly correlated with hematocrit-to-viscosity ratio (HVR) at the highest shear rates in the overall population at visit 4 (p<0.05). In addition, L-glutamine C max for SCD participants was associated with increased hemoglobin concentration, an effect that was observed at visit 4 (p=0.025), and increased %DRBC, an effect detected at visits 3 (p=0.008) and 4 (p=0.007). In PK-PD analysis of ROS, L-glutamine AUC was inversely correlated with ROS Retic (p=0.024), but not ROS RBC, in all participants at visit 3. When considered as total ROS Retic (calculated as ROS retic × absolute reticulocyte count), there was a similar inverse correlation with glutamine AUC in SCD participants, although not statistically significant. In contrast to all other biomarkers, L-glutamine C max was not correlated with any ROS measurement. This PK-PD analysis of L-glutamine reveals biological effects that alter RBC characteristics and could modify SCD-related complications, with a complex interplay summarized in Figure 1. Prospective studies can validate these biomarkers and be used to monitor the effects of L-glutamine therapy in SCD patients.

Heterogeneity of Red Blood Cell Deformability Caused by Lipopolysaccharide based on a Microfluidic Chip.

Alterations in red blood cell deformability (RBC-df) provide important information for the diagnosis of various diseases. We evaluated individual differences of lipopolysaccharide (LPS)-induced oxidative damage of RBC-df and analyzed the correlation between RBC-df and biochemical parameters. A microfluidic chip was developed to detect inter-individual variability of different concentrations of LPS-induced oxidative damage of RBC-df in 9 healthy volunteers. The relationships between various biochemical indicators (Na+-K+-ATPase activity, lipid peroxide (LPO) content, glutathione peroxidase (GSH-PX) activity, catalase (CAT) activity, superoxide dismutase (SOD) activity, adenosine triphosphate (ATP) content, and hemoglobin (HB) content) and RBCsdf were investigated. The obvious inter-individual variability of LPS-induced oxidative damage of RBC-df was revealed. The Na+-K+-ATPase activity, LPO content, GSH-PX activity, and CAT activity of RBCs showed significant correlations with RBC-df (P < 0.05). Oxidative damage and energy metabolism are the critical factors of RBC-df impairment induced by LPS, and the individual dependence on RBC-df is an important indicator for the treatment of infection-associated sepsis since antibiotics can kill pathogenic bacteria, which results in the release of LPS from the cell wall.

New erythrocyte parameters derived from the Coulter principle relate with red blood cell properties-A pilot study in diabetes mellitus.

In routine hematological instruments, blood cells are counted and sized by monitoring the impedance signals induced during their passage through a Coulter orifice. However, only signals associated with centered paths in the aperture are considered for analysis, while the rejected measurements, caused by near-wall trajectories, can provide additional information on red blood cells (RBC), as recent publications suggest. To assess usefulness of two new parameters in describing alterations in RBC properties, we performed a pilot study to compare blood samples from patients with diabetes mellitus (DM), frequent pathological condition associated with impairment in RBC deformability, versus controls. A total of 345 blood samples were analyzed: 225 in the DM group and 120 in the control group. A diagram of [Formula: see text] and [Formula: see text], the two new parameters derived from the analysis of impedancemetry pulses, was used to compare distribution of RBC subpopulations between groups. To discriminate RBC from DM and control individuals, based on our multiparametric analysis, we built a score from variables derived from [Formula: see text] matrix which showed good performances: area under the receiving operating characteristic curve 0.948 (0.920-0.970), p<0.0001; best discriminating value: negative predictive value 94.7%, positive predictive value was 78.4%. These results seem promising to approach RBC alterations in routine laboratory practice. The related potential clinically relevant outcomes remain to be investigated.

Low-Dose Dietary Fish Oil Improves RBC Deformability without Improving Post-Transfusion Recovery in Mice.

Long-chain polyunsaturated fatty acids (LC-PUFAs) are important modulators of red blood cell (RBC) rheology. Dietary LC-PUFAs are readily incorporated into the RBC membrane, improving RBC deformability, fluidity, and hydration. Female C57BL/6J mice consumed diets containing increasing amounts of fish oil (FO) ad libitum for 8 weeks. RBC deformability, filterability, and post-transfusion recovery (PTR) were evaluated before and after cold storage. Lipidomics and lipid peroxidation markers were evaluated in fresh and stored RBCs. High-dose dietary FO (50%, 100%) was associated with a reduction in RBC quality (i.e., in vivo lifespan, deformability, lipid peroxidation) along with a reduced 24 h PTR after cold storage. Low-dose dietary FO (6.25-12.5%) improved the filterability of fresh RBCs and reduced the lipid peroxidation of cold-stored RBCs. Although low doses of FO improved RBC deformability and reduced oxidative stress, no improvement was observed for the PTR of stored RBCs. The improvement in RBC deformability observed with low-dose FO supplementation could potentially benefit endurance athletes and patients with conditions resulting from reduced perfusion, such as peripheral vascular disease.